- Email: [email protected]
Structural domains in type I collagen and their role in OI
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
with miR-378 inhibited cell differentiation, confirming a role of endogenous NN. MC3T3 cells stably transfected with NN exhibited higher rates of differentiation and nodule formation than those transfected with NN containing the 3’-UTR in the early stages, suggesting the presence of endogenous miR-378. However, in the later stages, the differentiation rates were opposite, higher rates of differentiation and nodule formation in the 3’-UTR containing group. This appeared to be the consequence of competition between NN and GlcNAc transferase for miR-378, resulting in increased GlcNAc transferase activity, NN glycosylation and product secretion. doi:10.1016/j.matbio.2006.08.210
188 DNA methylation of collagen genes by RFX1 recruitment of DNMTs P.K. Sengupta, A. Agarwal, B.D. Smith Boston University School of Medicine, Boston, MA, United States Collagen type I transcriptional start sites are methylated in various cancer cell lines and human colon cancer tissue suggesting a role for collagen methylation in tumorigenesis. Regulatory factor for X-box 1 (RFX1) binds to the transcription start site of both type I collagen genes in a methylation sensitive manner and represses collagen transcription. DNA methylation is maintained by DNA methyltransferase 1 (DNMT1, maintenance methyltransferase) while de novo methylation is mediated by DNMT3 isoforms. Our results demonstrate that RFX1, co-immunoprecipitates with DNMTs containing substantial activity using unmethylated COL1A2 sequence and poly dI.dC as substrates. DNA affinity precipitation assay using biotin labeled methylated COL1A2 sequence (−25/+30 bp) and nuclear protein indicate that active DNMT is recruited to the collagen start site and can methylate unmethylated COL1A2 substrate. Transient transfection assays performed with collagen promoter in human fibroblast cells indicate that DNMT1 and DNMT3B can repress collagen promoter activity. HT1080 cells have a partially methylated collagen gene and collagen gene expression is repressed. Chromatin immunoprecipitation experiments using HT1080 chromatin demonstrate that RFX1 and all isoforms of DNMTs occupy the collagen start site. The occupancy of RFX1 and the DNMT decreases upon exposure to a 3 day treatment with different concentrations of the demethylating agent, 5-aza-2’-deoxycytidine (aza-dC). Our results indicate that spreading of methylation on the collagen gene in cancer may be mediated in part by the interaction of RFX1 and DNMT whereby RFX1 recruits DNMTs to the collagen start site. doi:10.1016/j.matbio.2006.08.211
S77
189 Structural domains in type I collagen and their role in OI E. Makareeva a, N.V. Kuznetsova a, M.B. Sutter a, W.A. Cabral b, J.C. Marini b, S. Leikin a a
Section on Physical Biochemistry, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States b Bone and Extracellular Matrix Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States By differential scanning calorimetry, circular dichroism, differential fluorescent labeling and other physical and biochemical techniques we screened over three dozen Osteogenesis Imperfecta mutations. We found that the effect of an obligate Gly substitution on the stability of full-length collagen depends on the location of the mutation within different structural domains, but not on the identity of the substitution or whether it is located within α1(I) or α2(I) chain. Regions of the triple helix which fold and unfold cooperatively are likely to play an important role in OI, e.g., a mutation causing a structural defect might affect the whole domain. Their existence was long suspected, but no specific domains were found before. The present study mapped a highly stable “N-anchor” domain containing the first 85 N-terminal amino acids of the triple helix and revealed evidence for at least few more such domains. Mutations in the N-anchor domain cause a distinct phenotype with combined OI and Ehlers-Danlos Syndrome (EDS) symptoms. The most flexible domain (∼aa 700-800) overlaps with one of major ligand binding regions. Glycine substitutions in α1(I) chain within this domain are predominantly lethal. Mutations within other structural domains might also give rise to distinct OI features. doi:10.1016/j.matbio.2006.08.212
190 Type XXVII collagen is made by mature hypertrophic chondrocytes J.M. Pace a, R. Hjorten a, U. Hansen b, R.A. Underwood a, R.J. Fernandes a , D. Krakow c, P. Bruckner b, R. Jacquet d, W.J. Landis d, P.H. Byers a a
University of Washington University of Muenster c UCLA d Northeastern Ohio Universities College of Medicine b
COL27A1 is a newly identified collagen gene and is expressed in cartilaginous tissue including the anlagen of endochondral
with miR-378 inhibited cell differentiation, confirming a role of endogenous NN. MC3T3 cells stably transfected with NN exhibited higher rates of differentiation and nodule formation than those transfected with NN containing the 3’-UTR in the early stages, suggesting the presence of endogenous miR-378. However, in the later stages, the differentiation rates were opposite, higher rates of differentiation and nodule formation in the 3’-UTR containing group. This appeared to be the consequence of competition between NN and GlcNAc transferase for miR-378, resulting in increased GlcNAc transferase activity, NN glycosylation and product secretion. doi:10.1016/j.matbio.2006.08.210
188 DNA methylation of collagen genes by RFX1 recruitment of DNMTs P.K. Sengupta, A. Agarwal, B.D. Smith Boston University School of Medicine, Boston, MA, United States Collagen type I transcriptional start sites are methylated in various cancer cell lines and human colon cancer tissue suggesting a role for collagen methylation in tumorigenesis. Regulatory factor for X-box 1 (RFX1) binds to the transcription start site of both type I collagen genes in a methylation sensitive manner and represses collagen transcription. DNA methylation is maintained by DNA methyltransferase 1 (DNMT1, maintenance methyltransferase) while de novo methylation is mediated by DNMT3 isoforms. Our results demonstrate that RFX1, co-immunoprecipitates with DNMTs containing substantial activity using unmethylated COL1A2 sequence and poly dI.dC as substrates. DNA affinity precipitation assay using biotin labeled methylated COL1A2 sequence (−25/+30 bp) and nuclear protein indicate that active DNMT is recruited to the collagen start site and can methylate unmethylated COL1A2 substrate. Transient transfection assays performed with collagen promoter in human fibroblast cells indicate that DNMT1 and DNMT3B can repress collagen promoter activity. HT1080 cells have a partially methylated collagen gene and collagen gene expression is repressed. Chromatin immunoprecipitation experiments using HT1080 chromatin demonstrate that RFX1 and all isoforms of DNMTs occupy the collagen start site. The occupancy of RFX1 and the DNMT decreases upon exposure to a 3 day treatment with different concentrations of the demethylating agent, 5-aza-2’-deoxycytidine (aza-dC). Our results indicate that spreading of methylation on the collagen gene in cancer may be mediated in part by the interaction of RFX1 and DNMT whereby RFX1 recruits DNMTs to the collagen start site. doi:10.1016/j.matbio.2006.08.211
S77
189 Structural domains in type I collagen and their role in OI E. Makareeva a, N.V. Kuznetsova a, M.B. Sutter a, W.A. Cabral b, J.C. Marini b, S. Leikin a a
Section on Physical Biochemistry, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States b Bone and Extracellular Matrix Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, United States By differential scanning calorimetry, circular dichroism, differential fluorescent labeling and other physical and biochemical techniques we screened over three dozen Osteogenesis Imperfecta mutations. We found that the effect of an obligate Gly substitution on the stability of full-length collagen depends on the location of the mutation within different structural domains, but not on the identity of the substitution or whether it is located within α1(I) or α2(I) chain. Regions of the triple helix which fold and unfold cooperatively are likely to play an important role in OI, e.g., a mutation causing a structural defect might affect the whole domain. Their existence was long suspected, but no specific domains were found before. The present study mapped a highly stable “N-anchor” domain containing the first 85 N-terminal amino acids of the triple helix and revealed evidence for at least few more such domains. Mutations in the N-anchor domain cause a distinct phenotype with combined OI and Ehlers-Danlos Syndrome (EDS) symptoms. The most flexible domain (∼aa 700-800) overlaps with one of major ligand binding regions. Glycine substitutions in α1(I) chain within this domain are predominantly lethal. Mutations within other structural domains might also give rise to distinct OI features. doi:10.1016/j.matbio.2006.08.212
190 Type XXVII collagen is made by mature hypertrophic chondrocytes J.M. Pace a, R. Hjorten a, U. Hansen b, R.A. Underwood a, R.J. Fernandes a , D. Krakow c, P. Bruckner b, R. Jacquet d, W.J. Landis d, P.H. Byers a a
University of Washington University of Muenster c UCLA d Northeastern Ohio Universities College of Medicine b
COL27A1 is a newly identified collagen gene and is expressed in cartilaginous tissue including the anlagen of endochondral